Francisella tularensis is a highly infectious bacterium responsible for tularemia, a disease whose pneumonic form has potentially lethal consequences in humans. Francisella virulence depends on its ability to survive and replicate inside macrophages of the infected host, where the bacterium down modulates the macrophage immune functions. The current model of Francisella intracellular fate is initial enclosure within a phagosome, followed by escape from this phagosome and then replication in the cytoplasm, but the bacterial determinants controlling these individual stages are unknown. We have been using cell biology-, bacterial genetics- and genomics-based approaches to further characterize Francisella intracellular trafficking, identify genes expressed at various stages of the intracellular cycle and assess their role in Francisella virulence. Using models of primary macrophage infection with F. tularensis, we have previously established the intracellular cycle of this pathogen, which involves rapid phagosomal escape (Checroun et al., 2006, PNAS, 103:14578; Chong et al., 2008, Infect. Immun., 76:5488) followed by extensive proliferation in the cytosol and autophagy-mediated reentry into the endocytic compartment at late stages of the cycle (Checroun et al., 2006, PNAS, 103:14578; Wehrly et al., 2009, Cell. Microbiol, 11:1128). In our efforts to understand how host factors modulate the Francisella intracellular cycle, we have established that targeting of Francisella to opsonic receptors via either complement or IgG opsonization, a process relevant to nave or immune hosts, negatively affects phagosomal escape and cytosolic proliferation, demonstrating that non-opsonized phagocytic pathways are more permissive to Francisella survival and proliferation than opsonic uptake processes (Geier and Celli, 2011, Infect. Immun., 79:2204). In our studies of the molecular mechanisms of Francisella intracellular pathogenesis, we have identified and characterized Francisella genes that are novel determinants of intracellular pathogenesis, through intracellular transcriptional profiling of the prototypical virulent strain Schu S4 of F. tularensis (Wehrly et al., 2009, Cell. Microbiol, 11:1128), among which two proteins encoded by these loci, DipA (FTT0369c) and FlpA (FTT1676), are surface-exposed during the intracellular cycle and required for cytosolic replication. The characterization of a dipA mutant has revealed that cytosolic replication-deficient Francisella eventually die within the macrophage cytosol and are cleared by autophagy, indicating that live bacteria must interfere with autophagic recognition during their cytosolic phase (Chong et al., 2012, Autophagy, in press). To test this hypothesis, we have generated and screened a library of transposon insertion mutants for clones that are efficiently targeted by autophagy rapidly after phagosomal escape in macrophages. We have identified several mutants in genetic loci showing functional convergence, indicating that this bacterium expresses specific factors that protect it against this major cytosolic innate immune process. Additionally, mutants in either dipA or flpA conferred high levels of protection of mice against a pulmonary challenge with a virulent strain, suggesting that such mutants have some potential as genetically defined, live vaccine strains against tularemia (Rockx-Brouwer et al., 2012, PLoS ONE, 10.1371/journal.pone.0037752).